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抗Ⅱ型登革病毒蛋白抗体的制备和特点分析
魏建春,崔晓云,吴娜,安静
首都医科大学基础医学院病原生物学系微生物学教研室,北京 100069
Preparation and characteristic analysis of anti­-dengue virus serotype 2 antibodies
WEI Jian-Chun, CUI Xiao-Yun, WU Na, AN Jing
Department of Microbiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China
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摘要: 本研究旨在制备和鉴定小鼠抗Ⅱ型登革病毒(DENV-2)10种蛋白的抗体,为后续相关研究提供实验材料。利用真核表达载体pReceiver构建DENV-2 10种蛋白的重组质粒,提取质粒DNA,肌内注射免疫小鼠,共免疫4次。末次免疫后2周取小鼠血清,利用DENV-2感染的Vero细胞和DENV-2各蛋白的稳定表达细胞,通过酶联免疫吸附试验(ELISA)、间接免疫荧光法(IFA)和蛋白免疫印迹法评价免疫效果,分析抗体的特点。DNA免疫小鼠后获得抗DENV-2 10种蛋白的抗血清,抗体效价波动于1:400~1:16 127之间,以抗E蛋白抗体效价最高,达1:16 127,而抗NS3、NS4b、NS5蛋白抗体效价较低,仅为1:400。利用DENV-2感染的Vero细胞和稳定表达病毒蛋白的EAhy926细胞进行IFA染色,抗DENV-2各蛋白的抗血清均可特异性识别DENV-2抗原。蛋白免疫印迹结果显示,抗E、NS1、NS4b和NS5蛋白抗体能识别热变性蛋白,其他抗血清未呈现阳性反应条带。本研究提示,DNA免疫小鼠所获得的抗DENV-2各蛋白抗体能特异性识别自然感染或模拟自然感染状态下的DENV-2蛋白,可为后续相关研究提供工具,也表明DNA免疫法可作为抗体制备的一种策略。

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魏建春
崔晓云
吴娜
安静
关键词 登革病毒结构蛋白非结构蛋白抗体EAhy926 细胞    
Abstract:Eukaryotic expression vector containing different proteins (C, M, E and NS1-­5) of dengue virus serotype 2 (DENV­-2) were constructed and intramuscularly injected directly to mice for developing antibodies against DENV-­2. Two weeks after the fourth immunization, sera were collected and the immune effects were evaluated by enzyme­linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and Western blotting. Specific antibodies could be obtained by DNA immunization, and the titers of these antibodies fluctuated between 1:400­ - 1:16 127. Of those, the antibody against protein E showed the highest titer at 1:16 127 whereas the antibodies against proteins NS3, NS4b and NS5 had the lowest titer at 1:400. The results showed that all antibodies acquired by DNA immunization could produce specific immunofluorescent reactions when tested using DENV­-2-infected Vero cells, suggesting that antibodies prepared by DNA immunization could specifically identify naturally infected DENV­-2. Antibodies against proteins E, NS1, NS4b and NS5 could also identify heat­denatured proteins. The results generated from this study provided the base for further research.

Key wordsDengue virus    Structure protein    Non-structure protein    Antibody    EAhy926 cell
基金资助:国家自然科学基金(81301435, 81401676, 81471957)

通讯作者: 安静   
引用本文:   
魏建春,崔晓云,吴娜,安静. 抗Ⅱ型登革病毒蛋白抗体的制备和特点分析[J]. 微生物与感染, 2015, 10(3): 147-153.
WEI Jian-Chun, CUI Xiao-Yun, WU Na, AN Jing. Preparation and characteristic analysis of anti­-dengue virus serotype 2 antibodies. JOURNAL OF MICROBES AND INFECTIONS, 2015, 10(3): 147-153.
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http://jmi.fudan.edu.cn/CN/     或     http://jmi.fudan.edu.cn/CN/Y2015/V10/I3/147

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